1. Field of the Invention
This invention relates to serum-free or serum-depleted culture media for supporting the proliferation and development of cells. More particularly, it is directed to culture media for the long-term growth of hematopoietic cells and the stromal cells which support their growth. It is also directed to methods for culturing mammalian cells from different tissues and organs using such media. The media and methods enable the maintenance of cell growth and development for up to several months.
II. Description of Background and Related Art
One aspect of the present invention is a medium for culturing cells, particularly mammalian cells in the absence of or substantial depletion of serum, whereby the medium is chemically well defined, while still providing long-term support of development and proliferation of such cells.
In the past, many types of mammalian cells have been isolated and attempts have been made to propagate the cells in culture for further study or use. The media used for such culture have typically included sources of nutrients, adhesion factors and growth factors required for cell proliferation and development, many provided by horse, bovine or calf serum. However, the use of serum to support cell culture has been problematic since it does not provide physiological conditions. Indeed, the only time a cell would see the sort of serum concentration normally used for cell culture would be at the site of a wound, where the blood is clotting. The use of serum is further complicated by high cost and the presence of undefined components in the serum which may vary from sample to sample. The presence or absence of these undefined components has led to inconsistent results in the culture of cells and lack of control over the culturing process. For example, a particular serum sample may introduce particular components into the culture medium that inhibit the growth of the cells.
In response to problems associated with the presence of serum, a variety of serum-free media have been produced in the past. See for example:
1) Barnes, D. & Sato, G. Serum- free cell culture. A unifying approach. Cell. 1980; 22: 649; PA1 2) Cormier, F., Ponting, I. L. O., Heyworth, C. M. & Dexter, T. M. Serum-free culture of enriched murine haemopoietic stem cells I: Effect of haemopoietic growth factors on proliferation. Growth Factors. 1991; 4: 157-164; PA1 3) Deslex, S., Negrel, R. & Ailhaud, G. Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells. Experimental Cell Research. 1987; 168: 15-30; PA1 4) Drouet, X., Douay, L., Giarratana, M. C., Baillou, C. L. Gorin, N. C., Salmon, C. H. & Najman, A. Human long-term bone marrow culture in serum-free medium. British Journal of Haematology. 1989; 73: 143-147; PA1 5) Kumar, R. K., O'Grady, R., Li, W., Smith, L. W., Rhodes, G. C. Primary culture of adult mouse lung fibroblasts in serum-free medium: responses to growth factors. Experimental Cell Research. 1991; 193:398-404; PA1 6) Migliaccio, G. Migliaccio, A. R. & Adamson, J. W. The biology of hematopoietic growth factors: Studies in vitro under serum-deprived conditions. Experimental Hematology 1990; 18: 1049-1055; and PA1 7) Ponting, I. L. O., Heyworth, C. M., Cormier, F. & Dexter, T. M. Serum-free culture of enriched murine haemopoietic stem cells II: Effects of growth factors and haemin on development. Growth Factors. 1991; 4: 165-173. PA1 (a) a standard culture medium such as Iscove's modified Dulbecco's medium (IMDM), RPMI, DMEM, Fischer's, alpha medium, Leibovitz's L-15, NCTC, F-10, MEM and McCoy's; PA1 (b) serum albumin; PA1 (c) transferrin; PA1 (d) a source of lipids and fatty acids; PA1 (e) cholesterol; PA1 (f) a reducing agent; PA1 (g) pyruvate; PA1 (h) nucleosides for synthesis of DNA and RNA; PA1 (i) one or more growth factors that stimulate the proliferation and development of stromal cells and/or cells from a variety of organs and tissues (preferable mammalian), such as epidermal growth factor, basic fibroblast growth factor, platelet derived growth factor, and insulin; and PA1 (j) one or more extracellular matrix materials; PA1 (k) a glucocorticoid such as hydrocortisone, cortisol, dexamethasone or other structurally related, natural or synthetic molecule. PA1 (1) stromal cells, which provide a supporting network for hematopoietic cells, as described below in greater detail.
As described in a number of the cited articles, there already exist methods for the short-term (up to 3-4 weeks) serum-free culture of hematopoietic progenitor cells, using added hematopoietic growth factors as the proliferative and developmental stimuli. However, the development of ideal serum-free media to study the long-term culture of hematopoietic cells has proven to be particularly difficult.
Prior to the invention described herein, mammalian long-term hematopoiesis has been studied in vitro mainly using serum supplemented bone marrow culture systems which appear to closely mimic some of the processes that occur in vivo. The first of these was described by Dexter, T. M., et al, J. Cell. Physiol., 91: 335-344 (1977) and involves the formation of an adherent layer of murine bone marrow stromal cells, which included endothelial cells, fibroblasts, adipocytes and macrophages. This cell layer was required to support the hematopoietic cells, probably by providing a physical adhesive matrix as well as the correct cell to cell signalling, including the necessary growth factors for hematopoietic cell proliferation and development.
The hematopoiesis which takes place in these Dexter cultures occurs in intimate contact with the stromal cell layer. In many cases the stem cells and progenitor cells proliferate under the stromal cells forming foci called cobblestone regions due to their characteristic appearance under phase microscopy. As the hematopoietic cells mature, many of them migrate to the uppermost surface of the stromal cells. Further maturation to a fully differentiated state results in release of the hematopoietic cells into the surrounding medium where they are removed by the bi-weekly feeding.
This culture system enabled the maintenance of hematopoiesis for several months, with the constant production of progenitor cells. Soon after, a similar system was developed for human cells (Gartner, S. and Kaplan, H. S., Proc. Natl. Acad. Sci. USA, 77: 4756-4759 (1980)).
One important drawback of the Dexter method was that it mainly allowed the development of macrophages and neutrophils, at the expense of other cell types normally produced by the bone marrow, particularly B lymphocytes. A method for the long-term production of B lymphocytes was discovered by Whitlock, C. A. and Witte, O. N., Proc. Natl. Acad. Sci. USA 79: 3608-3612 (1982). This system differed from that described by Dexter, et al. in that the horse serum was replaced with fetal calf serum and hydrocortisone was replaced with 2-mercaptoethanol. However, this system precluded the development of hematopoietic lineages other than B lymphocytes. The reason(s) for the differences in the development that occurs in these two culture systems, and why they do not mimic all aspects of bone marrow hematopoiesis, is unknown but is possibly a reflection of the use of serum.
In spite of the many attempts that have been made to produce an ideal, chemically controlled medium for culturing cells, especially mammalian hematopoietic cells, a number of shortcomings have persisted in such media. For example, prior media do not truly enable long-term culture of such cells. In accordance with the present invention, "long-term" is defined as greater than or equal to approximately eight weeks of continuous proliferation and development, including generation of new progenitor cells in the medium. The media of the present invention can produce up to three to five months of continuous growth and development in culture. There have been no prior serum-free media that have been able to achieve such long-term culture of mammalian hematopoietic cells.
The following is a brief description of some of the additional related prior art:
Cormier, et al. Growth Factors. 1991; 4: 157-164, relates to serum-free culture of enriched murine hematopoietic stem cells and focuses on the effect of certain growth factors on proliferation. However, the medium disclosed in this publication contains different components in different relative proportions as compared to the present invention, and was unable to achieve any long-term growth (as defined herein), in contrast to the present media.
Dexter, T. M., et al., J. Cell. Physiol. 91: 335-344 (1977), discloses the classical Dexter long-term bone marrow culture method. It is notable that this method contains serum, unlike the present invention. As a result its use is restricted to certain mouse strains and even with such strains the growth is not as pronounced as in the current invention.
Dexter, T. M., et al., Long-Term Bone Marrow Culture. New York: Alan R. Liss: 57-96, is a review of long-term marrow culture techniques and media. None of the techniques or media disclosed in this reference contain the same group of components as the present invention.
Drouet, X., et al. Brit. J. of Haem. 73: 143-147 (1989), disclose a human long-term bone marrow culture medium that is serum-free. However, the medium disclosed therein is not capable of achieving the same degree of long-term culture as in the present invention, growth being maintained for only 3-4 weeks. Furthermore, unlike the media of the present invention, those discussed by Drouet, et al. do not involve stromal cells.
Whitlock, C. A., & Witte, O.N. P.N.A.S. 79: 3608-3612 (1982), relates to long-term culture of B lymphocytes and their precursors from murine bone marrow. This is the famous Witte-Whitlock culture technique. However, again, the media disclosed in this paper are substantially different from those claimed in the present invention, in that they contain serum and can only support B lymphocyte growth.
Teofili, L., et al. Ann. Hematol. 65: 22-25 (1992), describes a serum-free culture system for the growth of human hematopoietic progenitor cells using a preformed extracellular matrix. However, this method does not produce long-term growth, progenitor cells only being maintained in reasonable numbers for 3 weeks. In addition, this method does not use growth factors to support the growth of the bone marrow stromal cells and does not use purified extracellular matrix materials for cell adherence. As a result it does not support the growth of cells from a wide variety of tissues and organs.
In spite of the above prior art, there remains a need to produce chemically defined media for long-term culture of cells. Ideally, such media should be simple to prepare, contain chemically defined ingredients, be economical to produce, and achieve optimal long-term growth and development of cells.
Accordingly, it is an object of the present invention to provide serum-free media for long-term maintenance of proliferation and development of cells, especially of hematopoietic origin.
A further object of the present invention is to provide methods for both short- and long-term culture of cells in a chemically defined medium.
Another object of this invention is to provide a method for stimulating the proliferation and/or development of early progenitor cells for bone marrow transplants and/or gene transfer into these cells for gene therapy procedures.
These and other objects of the invention as will hereinafter be described in greater detail will be apparent to one of ordinary skill in the art.